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Phenolic metabolism is rewired during internode development and in response to cinnamyl alcohol dehydrogenase perturbation in sorghum, with different sets of genes and accumulating compounds. The molecular mechanisms associated with secondary cell wall (SCW) deposition in sorghum remain largely uncharacterized. Here, we employed untargeted metabolomics and large-scale transcriptomics to correlate changes in SCW deposition with variation in global gene expression profiles and metabolite abundance along an elongating internode of sorghum, with a major focus on lignin and phenolic metabolism. To gain deeper insight into the metabolic and transcriptional changes associated with pathway perturbations, a bmr6 mutant [with reduced cinnamyl alcohol dehydrogenase (CAD) activity] was analyzed. In the wild type, internode development was accompanied by an increase in the content of oligolignols, p-hydroxybenzaldehyde, hydroxycinnamate esters, and flavonoid glucosides, including tricin derivatives. We further identified modules of genes whose expression pattern correlated with SCW deposition and the accumulation of these target metabolites. Reduced CAD activity resulted in the accumulation of hexosylated forms of hydroxycinnamates (and their derivatives), hydroxycinnamaldehydes, and benzenoids. The expression of genes belonging to one specific module in our co-expression analysis correlated with the differential accumulation of these compounds and contributed to explaining this metabolic phenotype. Metabolomics and transcriptomics data further suggested that CAD perturbation activates distinct detoxification routes in sorghum internodes. Our systems biology approach provides a landscape of the metabolic and transcriptional changes associated with internode development and with reduced CAD activity in sorghum.

Although cell wall polymers play important roles in the tolerance of plants to abiotic stress, the effects of salinity on cell wall composition and metabolism in grasses remain largely unexplored. Here, we conducted an in‐depth study of changes in cell wall composition and phenolic metabolism induced upon salinity in maize seedlings and plants. Cell wall characterization revealed that salt stress modulated the deposition of cellulose, matrix polysaccharides and lignin in seedling roots, plant roots and stems. The extraction and analysis of arabinoxylans by size‐exclusion chromatography, 2D‐NMR spectroscopy and carbohydrate gel electrophoresis showed a reduction of arabinoxylan content in salt‐stressed roots. Saponification and mild acid hydrolysis revealed that salinity also reduced the feruloylation of arabinoxylans in roots of seedlings and plants. Determination of lignin content and composition by nitrobenzene oxidation and 2D‐NMR confirmed the increased incorporation of syringyl units in lignin of maize roots. Salt stress also induced the expression of genes and the activity of enzymes enrolled in phenylpropanoid biosynthesis. The UHPLC–MS‐based metabolite profiling confirmed the modulation of phenolic profiling by salinity and the accumulation of ferulate and its derivatives 3‐ and 4‐O‐feruloyl quinate. In conclusion, we present a model for explaining cell wall remodeling in response to salinity.

As ever more component monomers are discovered, lignin can no longer be regarded as deriving from just the three canonical monolignols. Pathway intermediates and additional products of truncated biosynthesis are now established lignin monomers. The array of acylated monolignols continues to expand. Game-changing findings have demonstrated that phenolics from alternative pathways, including flavonoids and hydroxystilbenes, are also involved in lignification, expanding the traditional concept. Beyond the basic science intrigue, these findings propound exciting new avenues for valorizing lignins, or for producing more readily extractable or depolymerizable lignins, in crop and bioenergy plants.

MAIN CONCLUSION Lignin biosynthesis is uniquely malleable, allowing a variety of phenolics to be utilized as lignin monomers and therefore allowing some tailoring of its structure, reactivity, and value. We are now at a juncture where actual ‘design’ of the polymer can be contemplated and envisioning an ideal polymer for lignin valorization can be entertained. INTRODUCTION Evidence continues to mount regarding lignins’ inherent structural malleability from studies on lignin pathway mutants and transgenics as well as on various ‘natural’ plants discovered to possess unusual lignins. [1-4] Most of the monomers previously considered were from the monolignol biosynthetic pathway itself. More recently, phenolics from beyond the monolignol pathway have been shown to be authentic monomers in some plants, including the flavone tricin in all grasses (and beyond),[5,6] various hydroxystilbenes in some palm endocarp tissues,[7,8] and now possible nitrogenous compounds such a putrescine in maize kernel lignin. [9] As we have frequently noted from some time back, but not in print until 2008: “any phenolic transported to the lignifying zone of the cell wall can, subject to simple chemical compatibility, be incorporated into the polymer.”[10] That does not automatically mean that the r sulting polymer will be well tolerated by the plant, but researchers are now able to contemplate some degree of actually designing lignins for improved utilization and value, and muse over what might constitute a lignin that is ideally suited for conversion to phenolic monomers, adding value to the biorefinery. RESULTS AND DISCUSSION As José Carlos del Río will cover the other pathways that are now known to contribute to lignification, we shall provide an update to how lignins can be designed to fall apart more readily during processing (the so-called ‘zip-lignin’ approach) and describe a lignin that is natural in some tissues (but not yet in plant stems) that is one example of an ideal lignin. Zip, REFERENCES [1] Y. Mottiar, R. Vanholme, W. Boerjan, J. Ralph, S. D. Mansfield, Current Opinion in Biotechnology 2016, 37, 190. [2] R. Vanholme, K. Morreel, C. Darrah, P. Oyarce, J. H. Grabber, J. Ralph, W. Boerjan, New Phytologist 2012, 196, 978. [3] J. Ralph, Phytochemistry Reviews 2010, 9, 65. [4] W. Boerjan, J. Ralph, M. Baucher, Annual Review of Plant Biology 2003, 54, 519. [5] W. Lan, J. Rencoret, F. Lu, S. D. Karlen, B. G. Smith, P. J. Harris, J. C. del Rio, J. Ralph, The Plant Journal 2016, 88, 1046. [6] J. C. del Río, J. Rencoret, P. Prinsen, Á. T. Martínez, J. Ralph, A. Gutiérrez, Journal of Agricultural and Food Chemistry 2012, 60, 5922. [7] J. C. del Río, J. Rencoret, A. Gutiérrez, H. Kim, J. Ralph, Plant Physiology 2017, 174, 2072. [8] J. Rencoret, H. Kim, A. B. Evaristo, A. Gutiérrez, J. Ralph, J. C. del Río, Journal of Agricultural and Food Chemistry 2018, 66, 138. [9] J. C. del Río, J. Rencoret, A. Gutierrez, H. Kim, J. Ralph, Journal of Agricultural & Food Chemistry 2018, in press (accepted 4/20/2018). [10] J. Ralph, G. Brunow, P. J. Harris, R. A. Dixon, P. F. Schatz, W. Boerjan, in Recent Advances in Polyphenol Research, Vol. 1 (Eds.: F. Daayf, A. El Hadrami, L. Adam, G. M. Ballance), Wiley-Blackwell Publishing, Oxford, UK, 2008, pp. 36-66. [11] C. G. Wilkerson, S. D. Mansfield, F. Lu, S. Withers, J.-Y. Park, S. D. Karlen, E. Gonzales-Vigil, D. Padmakshan, F. Unda, J. Rencoret, J. Ralph, Science 2014, 344, 90. [12] S. Zhou, T. Runge, S. D. Karlen, J. Ralph, E. Gonzales-Vigil, S. D. Mansfield, ChemSusChem 2017, 10, 3565. [13] S. D. Karlen, C. Zhang, M. L. Peck, R. A. Smith, D. Padmakshan, K. E. Helmich, H. C. A. Free, S. Lee, B. G. Smith, F. Lu, J. C. Sedbrook, R. Sibout, J. H. Grabber, T. M. Runge, K. S. Mysore, P. J. Harris, L. E. Bartley, J. Ralph, Science Advances 2016, 2, e1600393. [14] F. Chen, Y. Tobimatsu, L. Jackson, J. Nakashima, J. Ralph, R. A. Dixon, The Plant Journal 2013, 73, 201. [15] F. Chen, Y. Tobimatsu, D. Havkin-Frenkel, R. A. Dix

Lignin is an abundant aromatic plant cell wall polymer consisting of phenylpropanoid units in which the aromatic rings display various degrees of methoxylation. Tricin [5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one], a flavone, was recently established as a true monomer in grass lignins. To elucidate the incorporation pathways of tricin into grass lignin, the metabolites of maize (Zea mays) were extracted from lignifying tissues and profiled using the recently developed ‘candidate substrate product pair’ algorithm applied to ultra-high-performance liquid chromatography and Fourier transform-ion cyclotron resonance-mass spectrometry. Twelve tricin-containing products (each with up to eight isomers), including those derived from the various monolignol acetate and p-coumarate conjugates, were observed and authenticated by comparisons with a set of synthetic tricin-oligolignol dimeric and trimeric compounds. The identification of such compounds helps establish that tricin is an important monomer in the lignification of monocots, acting as a nucleation site for starting lignin chains. The array of tricin-containing products provides further evidence for the combinatorial coupling model of general lignification and supports evolving paradigms for the unique nature of lignification in monocots. Lignin is one of the major components in plant cell walls and is deposited predominantly in the walls of secondarily thickened cells. It is a complex phenylpropanoid polymer composed primarily of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units derived from the monolignols p-coumaryl 2h, coniferyl 2g, and sinapyl 2s alcohols, respectively (Fig. 1; Freudenberg and Neish, 1968). These monolignols are biosynthesized in the cytoplasm and translocated to the cell wall, where they are oxidized by laccases and peroxidases to monolignol radicals (Boerjan et al., 2003; Dixon and Reddy, 2003; Ralph et al., 2004b; Vanholme et al., 2008, 2010; Bonawitz and Chapple, 2

Black poplar (Populus nigra L.) is a keystone species for riparian ecosystems in Europe. We analysed the structure of genetic diversity of 17 populations from 11 river valleys that are part of seven catchment systems (Danube, Ebro, Elbe, Po, Rhine, Rhone, and Usk) in Europe, in relation to geography and river management. In total, 1069 trees were genotyped using AFLP and microsatellite markers. The trees had an observed heterozygosity of 0.74 (range 0.59-0.82 across microsatellite loci). The majority (72.6-90.8%, depending on the marker system) of the genetic variation was present within populations. Most pairs of populations along a river were relatively similar (pairwise Fst 0.042-0.135 based on AFLP, 0.002-0.037 based on microsatellites). Overall population differentiation among rivers was considerable (Fst among populations was 0.268 based on AFLP, and 0.081 based on microsatellites). An analysis using the program Structure indicated that all populations recruited plants from several clusters. Geographically close populations tended to draw from the same Structure clusters, including populations from adjacent catchments. The Danube and Inn populations in Austria were genetically more similar to the Vltava population (Elbe catchment) in Czech Republic than the geographically more distant populations along the Tisa and Prut rivers of the Danube catchment in Ukraine. This indicates that gene flow and dispersal takes place across fairly large distances and between river catchments. Consistent with this result, a principal coordinate analysis of genetic distances among individual trees based on AFLP bands showed large overlap of populations, although the French and Spanish samples formed distinct clusters, and the samples from the Ticino (Italy) were at an intermediate position. The extent of clonal duplication was highest along regulated rivers, with e.g., 41% clonal duplication along the Rhine in The Netherlands (up to 32 trees for one genet). The Usk contained a m

Populus nigra L. is a pioneer tree species of riparian ecosystems that is threatened with extinction because of the loss of its natural habitat. To evaluate the existing genetic diversity of P. nigra within ex-situ collections, we analyzed 675 P. nigra L. accessions from nine European gene banks with three amplified fragment length polymorphism (AFLP) and five microsatellite [or simple sequence repeat (SSR)] primer combinations, and 11 isozyme systems. With isozyme analysis, hybrids could be detected, and only 3% were found in the gene bank collection. AFLP and SSR analyses revealed effectively that 26% of the accessions were duplicated and that the level of clonal duplication varied from 0% in the French gene bank collection up to 78% in the Belgian gene bank collection. SSR analysis was preferred because AFLP was technically more demanding and more prone to scoring errors. To assess the genetic diversity, we grouped material from the gene banks according to topography of the location from which the accessions were originally collected (river system or regions separated by mountains). Genetic diversity was expressed in terms of the following parameters: percentage of polymorphic loci, observed and effective number of alleles, and Nei's expected heterozygosity or gene diversity (for AFLP). Genetic diversity varied from region to region and depended, to some extent, on the marker system used. The most unique alleles were identified in the Danube region (Austria), the Rhône region (France), Italy, the Rijn region (The Netherlands), and the Ebro region (Spain). In general, the diversity was largest in the material collected from the regions in Southern Europe. Dendrograms and principal component analysis resulted in a clustering according to topography. Material from the same river systems, but from different countries, clustered together. The genetic differentiation among the regions (Fst/Gst) was moderate. © Springer-Verlag 2003.

© 2014 American Society of Plant Biologists. All Rights Reserved. Tricinwas recentlydiscoveredinligninpreparations fromwheat (Triticum aestivum) strawandsubsequentlyinallmonocotsamplesexamined. To provide proof that tricin is involved in lignification and establish the mechanism bywhich it incorporates into the lignin polymer, the 4¢-O-b coupling products of tricinwith themonolignols (p-coumaryl, coniferyl, and sinapyl alcohols) were synthesized alongwith the trimer that would result fromits 4¢-O-b-couplingwith sinapyl alcohol and then coniferyl alcohol. Tricinwas also found to cross couplewithmonolignols to form tricin-(4¢-O-b)-linked dimers in biomimetic oxidations using peroxidase/hydrogen peroxide or silver (I) oxide. Nuclear magnetic resonance characterization of gel permeation chromatography-fractionated acetylated maize (Zeamays) lignin revealed that the tricinmoieties are found in even the highestmolecularweight fractions, ether linked to lignin units, demonstrating that tricin is indeed incorporated into the lignin polymer.Thesefindingssuggest that tricin is fullycompatiblewithlignification reactions, isanauthentic ligninmonomer, and,because it can only start a lignin chain, functions as a nucleation site for lignification in monocots. This initiation role helps resolve a long-standing dilemma that monocot lignin chains do not appear to be initiated by monolignol homodehydrodimerization as they are in dicots that have similar syringyl-guaiacyl compositions. The term flavonolignin is recommended for the racemic oligomers and polymers ofmonolignols that start from tricin (or incorporate other flavonoids) in the cell wall, in analogy with the existing term flavonolignan that is used for the lowmolecular mass compounds composed of flavonoid and lignan moieties.

The global demand for food, feed, energy and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

In order to examine the potential for coproduct generation, we have characterised chemical compounds released by a range of alkaline and acidic aqueous pretreatments as well as the effect of these pretreatments on the saccharification ability of the lignocellulosic material. Comparative experiments were performed using three biomass types chosen for their potential as second-generation biofuel feedstocks: maize stover, miscanthus and sugarcane bagasse. The release of lignin from the feedstock correlated with the residual biomass saccharification potential, which was consistently higher after alkaline pretreament for all three feedstock types. Alkaline pretreatment released more complex mixtures of pentose and hexose sugars into the pretreatment liquor than did acid pretreatment. In addition, complex mixtures of aromatic and aliphatic compounds were released into pretreatment liquors under alkaline conditions, in a temperature-dependent manner, but far less so under acidic conditions. We show that the three feedstocks characterised interact with the pretreatment conditions in a specific manner to generate different ranges of products, highlighting the need to tailor pretreatments to both the starting feedstock and desired outcomes.